Difference between revisions of "Part:BBa K274003:Experience"

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===Applications of BBa_K274003===
 
===Applications of BBa_K274003===
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===User review of SHSBNU_China===
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Before our marine <i>Shewanella baltica</i> TtrS/R system, the only known genetically encoded tetrathionate sensor is the <i>Salmonella typhimurium</i> TtrS/R two-component system (TCS) (Hensel et al, 1999; Price-Carter et al, 2001). However, they found <i>S.typhimurium’s</i> promoter PttrB is repressed by oxygen and nitrate via the global regulator Nitrate Reductase Regulator (FNR). While the oxygen levels in the intestinal are poorly understood by human beings Kristina et al think the oxygen level maybe relatively high near the epithelial mucosal boundary because of near to the blood. Meanwhile, nitrate levels in intestinal have been shown to be improved during inflammation (Winter et al, 2013). In iGEM 2012, Dundee used <i>Salmonella</i> TtrS/R system and the system works well.
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The sensor we use <i>S.baltica</i> TtrS/R which is only weakly repressed by oxygen and not repressed by nitrate in <i>E. coli.</i> And our <strong><i>S.baltica</i> TtrS/R system can work well using protoviolaceinic acid as the reporter</strong>,which display dark-green color. And our system could also work poorly using sfGFP as the reporter.
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[[File: SHSBNU 17 40aL1.jpg|600px|thumb|center|Figure 1]]
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Figure 1 TtrS/R system and ThsS/R system
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ThsS (BBa_K2507000) and ThsR (BBa_K2507001), both codon-optimized for E. coli, are two basic parts which belong to the two-component system from the marine bacterium Shewanella halifaxensis.
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By linking thsR with chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or the violacein producing operon vioABDE (BBa_K2507012), this system can respond to thiosulfate by producing a signal visible to the naked eye, such as chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or a dark-green small-molecule pigment (protoviolaceinic acid).
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E. coli-codon-optimized TtrS(BBa_K2507002) and TtrR (BBa_K2507003) are two basic parts which are derived from the two-component system of the marine bacterium Shewanella baltica.
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By linking ttrR with chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or the violacein producing operon vioABDE (BBa_K2507012), this system can respond to thiosulfate by producing a signal visible to the naked eye such as chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or a dark-green small-molecule pigment (protoviolaceinic acid).
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[[File: SHSBNU 17 40aL2.jpg|600px|thumb|center|Figure 2]]
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Figure 2. Characterization of the ThsS/R and TtrS/R system by observing the chromprotein expression levels. We added 1mM, 0.1mM, 0.01mM and 0 Na2S2O3 to ThsS/R system and added 2.5mM, 1mM, 0.1mM and 0 Na2S4O6·2H2O to ThsS/R system. The results demonstrate there is an obvious response in the ThsS/R system. In a. gfasPurple system, the response curve is obvious, and b. spisPink & c. amilCP with rather heavy leaky expression without inducer. d.e.f. However, in TtrS/R system there is no clear result.
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[[File: SHSBNU 17 40aL3.jpg|600px|thumb|center|Figure 3]]
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Figure 3.
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Characterization of the ThsS/R and TtrS/R system by observing the protoviocaceinic acid( dark-green pigment). We added 1mM, 0.1mM, 0.01mM and 0 Na2S2O3 to ThsS/R system and added 2.5mM, 1mM, 0.1mM and 0 Na2S4O6·2H2O to ThsS/R system. The results demonstrate there is an obvious response in the ThsS/R system with rather heavy leaky expression without inducer. And the Ths/R+protoviolaceinic acid works rather well!
  
 
===User Reviews===
 
===User Reviews===
  
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E.coli turned dark green after transformation of this plasmid, it is not supposed to be so since there is no promoter upstream of the operon. We have not found the reason.
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E. coli turned dark green after transformation of this plasmid. This is unexpected as there is no promoter upstream of the operon.  
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BBa_K274003 Review No.2
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<I>UCL_London, Xiang Chen</I>
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Diagnostic DNA gel was run to test all 4 restriction sites. BBa_K274003 was cut with XbaI, PstI, EcoRI & SpeI, XbaI & PstI. A gel picture is shown as below.
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This plasmid did not cut with Xba I restriction site in our hands. As such we could not  use it to assemble after other parts.
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[[Image:K274003 Diagnostic Gel .jpg|center|300px]]
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|}
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== '''Characterization by 2012 UCSF iGEM Team ''' ==
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<html>
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<regulartext1>
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We also attempted to use this part as a PCR template and were unsuccessful. We obtained a plasmid containing these enzymes from another source (Dueber Lab, UC Berkeley) and have re-deposited this part (in a new form) with the registry as
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<br>
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- Part: <a href=https://parts.igem.org/Part:BBa_K726016>BBa_K726016</a></regulartext> <BR>
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<p>
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The purpose of this part is to produce prodeoxyviolaceinic acid, a green pigment. However, only the genes for producing the enzymes VioA, VioB, and VioE are necessary to produce the green pigment. Therefore, we have deposited our part (lacking VioD) as a smaller and improved version of BBa K274003. Additionally, BBa_K726016 has a T7 promoter and we have shown that it is able to make the green pigment:<br>
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<img align="center" style="margin-bottom:0px; width: 200px; margin-top:20px; padding:2; margin-left:55px;" src="https://dl.dropbox.com/u/24404809/iGEM%202012/igem%202012%20website%20photos/violacein/VioABE_Culture.jpg"> <br>
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</html>
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== '''Characterisation by Wits South Africa ''' ==
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We attempted to try and use the Dark Green E.chromi biobrick (BBa_K274003) in our machine constructs. Although the part was excised using EcoR1 and Spe1 (Fermentas), when we attempted to clone anything in front of this part, by digestion with EcoR1 and Xba1 (Fermentas), we could not obtain any positive clones.
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Suspecting that perhaps the restriction sites were not present, we tested the plasmid samples through restriction digests.
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[[image: wits_xbal_digest.jpg|400px]]
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'''Figure 1. E.chromi Dark Green digested with various restriction enzymes to confirm the presence of the Biobrick restriction sites.'''
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As can be noted from Figure 1, the Xba1 site is not present in the Dark Green E.chromi Biobrick part, although the Spe1 and EcoR1 sites are present.
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Latest revision as of 18:24, 1 November 2017

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K274003

User review of SHSBNU_China

Before our marine Shewanella baltica TtrS/R system, the only known genetically encoded tetrathionate sensor is the Salmonella typhimurium TtrS/R two-component system (TCS) (Hensel et al, 1999; Price-Carter et al, 2001). However, they found S.typhimurium’s promoter PttrB is repressed by oxygen and nitrate via the global regulator Nitrate Reductase Regulator (FNR). While the oxygen levels in the intestinal are poorly understood by human beings Kristina et al think the oxygen level maybe relatively high near the epithelial mucosal boundary because of near to the blood. Meanwhile, nitrate levels in intestinal have been shown to be improved during inflammation (Winter et al, 2013). In iGEM 2012, Dundee used Salmonella TtrS/R system and the system works well.

The sensor we use S.baltica TtrS/R which is only weakly repressed by oxygen and not repressed by nitrate in E. coli. And our S.baltica TtrS/R system can work well using protoviolaceinic acid as the reporter,which display dark-green color. And our system could also work poorly using sfGFP as the reporter.

Figure 1

Figure 1 TtrS/R system and ThsS/R system ThsS (BBa_K2507000) and ThsR (BBa_K2507001), both codon-optimized for E. coli, are two basic parts which belong to the two-component system from the marine bacterium Shewanella halifaxensis. By linking thsR with chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or the violacein producing operon vioABDE (BBa_K2507012), this system can respond to thiosulfate by producing a signal visible to the naked eye, such as chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or a dark-green small-molecule pigment (protoviolaceinic acid). E. coli-codon-optimized TtrS(BBa_K2507002) and TtrR (BBa_K2507003) are two basic parts which are derived from the two-component system of the marine bacterium Shewanella baltica.

By linking ttrR with chromoprotein genes (BBa_K2507009, BBa_K2507010, BBa_K2507011) or the violacein producing operon vioABDE (BBa_K2507012), this system can respond to thiosulfate by producing a signal visible to the naked eye such as chromoproteins (spisPink-pink chromoprotein, gfasPurple-purple chromoprotein, amilCP-blue chromoprotein) or a dark-green small-molecule pigment (protoviolaceinic acid).

Figure 2

Figure 2. Characterization of the ThsS/R and TtrS/R system by observing the chromprotein expression levels. We added 1mM, 0.1mM, 0.01mM and 0 Na2S2O3 to ThsS/R system and added 2.5mM, 1mM, 0.1mM and 0 Na2S4O6·2H2O to ThsS/R system. The results demonstrate there is an obvious response in the ThsS/R system. In a. gfasPurple system, the response curve is obvious, and b. spisPink & c. amilCP with rather heavy leaky expression without inducer. d.e.f. However, in TtrS/R system there is no clear result.

Figure 3

Figure 3. Characterization of the ThsS/R and TtrS/R system by observing the protoviocaceinic acid( dark-green pigment). We added 1mM, 0.1mM, 0.01mM and 0 Na2S2O3 to ThsS/R system and added 2.5mM, 1mM, 0.1mM and 0 Na2S4O6·2H2O to ThsS/R system. The results demonstrate there is an obvious response in the ThsS/R system with rather heavy leaky expression without inducer. And the Ths/R+protoviolaceinic acid works rather well!

User Reviews

E. coli turned dark green after transformation of this plasmid. This is unexpected as there is no promoter upstream of the operon.

BBa_K274003 Review No.2 UCL_London, Xiang Chen

Diagnostic DNA gel was run to test all 4 restriction sites. BBa_K274003 was cut with XbaI, PstI, EcoRI & SpeI, XbaI & PstI. A gel picture is shown as below.

This plasmid did not cut with Xba I restriction site in our hands. As such we could not use it to assemble after other parts.


K274003 Diagnostic Gel .jpg


Characterization by 2012 UCSF iGEM Team

We also attempted to use this part as a PCR template and were unsuccessful. We obtained a plasmid containing these enzymes from another source (Dueber Lab, UC Berkeley) and have re-deposited this part (in a new form) with the registry as
- Part: BBa_K726016

The purpose of this part is to produce prodeoxyviolaceinic acid, a green pigment. However, only the genes for producing the enzymes VioA, VioB, and VioE are necessary to produce the green pigment. Therefore, we have deposited our part (lacking VioD) as a smaller and improved version of BBa K274003. Additionally, BBa_K726016 has a T7 promoter and we have shown that it is able to make the green pigment:

Characterisation by Wits South Africa

We attempted to try and use the Dark Green E.chromi biobrick (BBa_K274003) in our machine constructs. Although the part was excised using EcoR1 and Spe1 (Fermentas), when we attempted to clone anything in front of this part, by digestion with EcoR1 and Xba1 (Fermentas), we could not obtain any positive clones.

Suspecting that perhaps the restriction sites were not present, we tested the plasmid samples through restriction digests.


Wits xbal digest.jpg

Figure 1. E.chromi Dark Green digested with various restriction enzymes to confirm the presence of the Biobrick restriction sites.


As can be noted from Figure 1, the Xba1 site is not present in the Dark Green E.chromi Biobrick part, although the Spe1 and EcoR1 sites are present.



UNIQec1508b02631316e-partinfo-00000001-QINU UNIQec1508b02631316e-partinfo-00000002-QINU